Fig 2: Conceptual figure illustrating the proposed impact of EDC exposure on the peri-pubertal epigenome and subsequent effects leading to changes in pubertal timing. High urinary levels of phthalates, presumably caused by higher exposure, were shown to be associated with higher promoter methylation of the TRIP6 promoter and lower circulating levels of TRIP6 protein. We have earlier shown that lower circulating levels of TRIP6 protein were associated with later pubertal onset. Also, higher urinary phthalate levels have earlier been shown to be directly associated with later pubarche and menarche (5, 6, 35, 36).

Fig 3: Identification of differentially methylated regions associated with pubertal age.(a) The most significant differentially methylated genomic region (nine probes with a mean p-value of 1.3e–31) was found on chromosome 7 (chr7:100463206-100464771) that contains both gene body and 3' UTR of SLC12A9 and transcription start site of TRIP6. The CpGs were located in several putative transcription factor bindings sites upstream of TRIP6. TX Factor ChIP, DNase1 clusters tracks were obtained from the ENCODE database and the CpG Islands from the UCSC database. (b) Immunohistochemical staining for TRIP6 in normal adult testicular tissue showing intense and specific staining in Leydig cells (arrow heads) producing testosterone. (c) TRIP6 staining of pre-pubertal (7.9 years old) testicular tissue. TRIP6 staining was absent from Leydig cells (arrow heads) in pre-pubertal testis. Bar equals 20 µm. Negative controls are shown in Supplementary Figure 4 together with staining of ovarian tissue. (d) Circulating levels of TRIP6 were determined by ELISA in boys and girls pre-pubertally, around pubertal onset and post-pubertally. The box plots show the distribution of the measurements (the band inside the box depicts the median) at each time point and the connected lines are drawn from the mean of each group. **Denotes a p-value below 0.01.

Fig 4: Overlap with the Comparative Toxicogenomics Database (CTD) and core protein–protein interacting network. Venn plots show the overlap between genes with promoter methylation associated with urinary levels of EDCs and genes known to be interacting with the EDCs as listed in the CTD. (A) TCS, (B) BP-3, (C) 2,4-DCP, (D) BPA, and (E) Sphth.m. Intersecting promoters are listed below each Venn plot and promoters also identified as overlapping between several EDCs (BAALC, CLDN9, FAM71F1, FAM83A, PNOC, and TRIP6) and promoters also identified between same-day EDC levels and the mean of yearly dichotomized levels of EDCs (BAALC, DTX1, FAM71F1, FBXO47, HES6, SLC38A4, SLC45A4, SOX10, TAPBP, and TRIP6) are highlighted in bold font. (F) All genes identified as having promoter methylation associated with EDC levels were used to search the STRING database for high confident protein–protein interactions. This identified a single core network of 63 proteins (small networks of six or less proteins were excluded), which included 27 genes previously identified to be shared in the different analyses (Figs 2 and 3A, B, C, D, E) and are marked with bold font. The core network also included nine proteins with well-known functions in pubertal development and the HPG-axis (e.g. BMP4, NFKB1, SOX10, TGFB2, and TRIP6) and these are marked with red font. Note that five proteins are both in red and bold font (BMP4, ISL1, SOX10, GNAS, and TRIP6) indicating that they both have promoter methylation associated with EDCs and have been described in relation to puberty.